The RNA editing ADAR enzymes convert adenosines to inosines in RNA. This phenomenon is widespread with > 15,000 A to I sites known in human transcripts. Since inosine is decoded as guanosine during translation, this modification can lead to changes in the meaning of codons (recoding). There are at least 50 different A to I sites in human mRNAs that cause recoding and recoding is common in the nervous system. Indeed, ADARs are necessary for a properly functioning nervous system and are known to regulate behavior in metazoans. Mutations in the human ADAR1 gene cause the skin disorder Dyschromatosis Symmetrica Hereditaria (DSH) and the autoimmune disease Aicardi-Goutieres Syndrome (AGS). Also, hyper editing has been observed at certain sites in cancer cells, such as in the mRNA for AZIN1 (antizyme inhibitor 1). In contrast however, hypo editing is observed in the glioma- associated ongene 1 (Gli1), a transcription factor important in the Hedgehog signaling pathway considered a therapeutic target in several types of cancer. Despite the significance of this form of regulation of RNA structure and function, our understanding of the mechanism of A to I editing is deficient. For instance, the selectivity for specific adenosines within ADAR substrates remains difficult to fully explain. Furthermore, pharmacological methods for controlling RNA editing at specific sites do not currently exist. In this competitive renewal o an R01 project, we will address these knowledge gaps through the application of nucleic acid chemistry coupled with techniques from structural biology and biochemistry. The results of these studies will extend our basic understanding of the process of RNA editing as well as lead to new methods for its control. Given our laboratory's recent discovery of small RNAs that react rapidly with isolated ADAR deaminase domains, we are in a position like never before to provide important information about the ADAR-RNA interaction. Thus, we will pursue X-ray diffraction quality crystals of ADAR deaminase domains in complex with RNAs derived from sequences shown by us to react rapidly and solve the structures in collaboration with Professor Andrew Fisher. We expect the resulting structures to reveal details of the ADAR-RNA interaction that will shed light on the observed editing site selectivity. In addition, we will directly test two competig hypotheses for the basis of editing site selectivity. In addition, to advance our goal of developin methods to control editing pharmacologically, we will generate new antisense oligonucleotides capable of regulating RNA editing in a site-specific manner (editing inhibitors and editing enhancers). The resulting compounds will be useful tools to probe the mechanism of the editing reaction, the biological function of specific editing events and could have long- term therapeutic potential. Finally, we will provide a deeper understanding of the ADAR reaction by identifying sites in the catalytic domain important for catalysis not previously appreciated by carrying out directed evolution to convert the hADAR3 catalytic domain into a functional RNA editing enzyme.